Unusually Situated Binding Sites for Bacterial Transcription Factors Can Have Hidden Functionality

A commonly accepted paradigm of molecular biology is that transcription factors control gene expression by binding sites at the 5' end of a gene. However, there is growing evidence that transcription factor targets can occur within genes or between convergent genes. In this work, we have investigated one such target for the cyclic AMP receptor protein (CRP) of enterotoxigenic Escherichia coli. We show that CRP binds between two convergent genes. When bound, CRP regulates transcription of a small open reading frame, which we term aatS, embedded within one of the adjacent genes. Our work demonstrates that non-canonical sites of transcription factor binding can have hidden functionality

The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae

Many bacteria use population density to control gene expression via quorum sensing. In Vibrio cholerae, quorum sensing coordinates virulence, biofilm formation, and DNA uptake by natural competence. The transcription factors AphA and HapR, expressed at low and high cell density respectively, play a key role. In particular, AphA triggers the entire virulence cascade upon host colonisation. In this work we have mapped genome-wide DNA binding by AphA. We show that AphA is versatile, exhibiting distinct modes of DNA binding and promoter regulation. Unexpectedly, whilst HapR is known to induce natural competence, we demonstrate that AphA also intervenes. Most notably, AphA is a direct repressor of tfoX, the master activator of competence. Hence, production of AphA markedly suppressed DNA uptake; an effect largely circumvented by ectopic expression of tfoX. Our observations suggest dual regulation of competence. At low cell density AphA is a master repressor whilst HapR activates the process at high cell density. Thus, we provide deep mechanistic insight into the role of AphA and highlight how V. cholerae utilises this regulator for diverse purposes

cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization

The bacterium Vibrio cholerae is native to aquatic environments and can switch lifestyles to cause disease in humans. Lifestyle switching requires modulation of genetic systems for quorum sensing, intestinal colonization, and toxin production. Much of this regulation occurs at the level of gene expression and is controlled by transcription factors. In this work, we have mapped the binding of cAMP receptor protein (CRP) and RNA polymerase across the V. cholerae genome. We show that CRP is an integral component of the regulatory network that controls lifestyle switching. Focusing on a locus necessary for toxin transport, we demonstrate CRP-dependent regulation of gene expression in response to host colonization. Examination of further CRP-targeted genes reveals that this behavior is commonplace. Hence, CRP is a key regulator of many V. cholerae genes in response to lifestyle changes.Cholera is an infectious disease that is caused by the bacterium Vibrio cholerae. Best known for causing disease in humans, the bacterium is most commonly found in aquatic ecosystems. Hence, humans acquire cholera following ingestion of food or water contaminated with V. cholerae. Transition between an aquatic environment and a human host triggers a lifestyle switch that involves reprogramming of V. cholerae gene expression patterns. This process is controlled by a network of transcription factors. In this paper, we show that the cAMP receptor protein (CRP) is a key regulator of V. cholerae gene expression in response to lifestyle changes

The <i>aatS</i> mRNA contains a functional ribosome binding site.

<p>The graph shows activity of different <i>aatS</i>:<i>lacZ</i> translational fusions. The wildtype ribosome binding site (5'-AAGAAG-3') in the <i>aatS</i>1 fragment was mutated to (5'-TTCTTC-3') in <i>aatS</i>2. LacZ activites was determined using the lysates of stationary phase M182 or M182<i>Δcrp</i>. In M182 cells <i>crp</i> was supplied in trans by plasmid pCRP that encodes <i>crp</i> under the control of its own promoter. Values shown are percentages of activity observed in strain M182 (5 Miller units). We obtained 0.25 and 0.26 Miller units of activity from lysates of M182 or M182Δ<i>crp</i>, carrying promoterless pRW225, respectively. Error bars represent the standard deviation of three independent experiments.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Characterisation of the P<i>aatS</i> promoter.

<p><b>A.</b> Primer extension analysis of the <i>aatS</i> transcript. Lanes 1–4 on the gel are arbitrary size standards, used for calibration, generated by sequencing of M13mp18 phage DNA. Lane 5 shows the primer extension product generated using RNA from wildtype M182 cells carrying the <i>aatS</i>1::<i>lacZ</i> fusion. Lane 6 shows the primer extension product generated using RNA from M182<i>Δcrp</i> cells carrying the <i>aatS</i>1::<i>lacZ</i> fusion. The transcription start site is indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157016#pone.0157016.g001" target="_blank">Fig 1B</a>. <b>B.</b> β-galactosidase activity determined using lysates of M182 wildtype or M182Δ<i>crp</i> cells carrying P<i>aatS</i> cloned upstream of <i>lacZ</i> in plasmid pRW50. Values shown are percentages of activity observed in strain M182 (92 Miller units). We obtained 7 and 3 Miller units of activity from lysates of M182 or M182Δ<i>crp</i> carrying promoterless pRW50. Error bars represent the standard deviation of three independent experiments. <b>C.</b> Multi-round i<i>n vitro</i> transcription assay using P<i>aatS</i>. The <i>aatS</i>1 DNA fragment was cloned into pSR upstream of a <i>λoop</i> terminator. Purified, supercoiled pSR plasmid was incubated with purified CRP at 37°C, and the reaction started by the addition of 400 nM σ⁷⁰- RNA polymerase holoenzyme. CRP concentrations are; 0 nM, 200 nM, or 400 nM. The 108 nt RNAI transcript from the pSR replication origin, and the 169 nt transcript from P<i>aatS</i>, are indicated. The gel is calibrated with an arbitrary G+A DNA sequencing reaction as a size standard.</p

The <i>aatPABC</i> operon of ETEC H10407.

<p>Schematic of the <i>aatPABC</i> operon and adjacent <i>tnpA</i> gene. The two DNA strands are shown as black lines. Known genes are shown as black arrows and the predicted <i>aatS</i> gene as a grey arrow. Gene names are shown in italic and gene function in parenthesis. The position of a putative CRP binding site is indicated by striped ovals. <b>A.</b> Sequence if the <i>tnpA</i>-<i>aatC</i> intergenic region. The CRP site is highlighted as a striped rectangle with the two half sites highlighted bold. The start codon of the <i>aatS</i> open reading frame is highlighted with a grey rectangle. The transcription start site, as determined by mRNA primer extension is denoted “+1” and indicated by a bent arrow. Distances upstream (-) and downstream (+) of this start site are numbered. The -35 and -10 hexamers are boxed, and the ribosome binding site (RBS) is underlined.</p